Considerable data associate transforming growth factor (TGF)-beta with glomerulosclerosis, but little is known about the intracellular signals that mediate TGF-beta effects. We propose to investigate possible fibrogenic roles for two major signal transduction pathways. TGF-betas activate at least one classical pathway, the mitogen activated protein (MAP) kinase cascade, that may mediate activation of ECM genes. A second potentially relevant pathway involves a novel, TGF-beta family-specific group of signaling proteins, the SMADs. Limited data are available regarding ligand-dependent activation of endogenous SMAD proteins, with no published data on the functional activity of these proteins in renal cell systems. Our preliminary results suggest that in human mesangial cells (l) TGF-beta1 stimulates collagen gene activation, (2) SMAD proteins are expressed and rapidly phosphorylated by TGF-beta1 treatment, (3) the ERK and JNK MAP kinases are stimulated by TGF-beta1, (4) inhibition of ERK activation decreases TGF-beta1-stimulated collagen mRNA expression (5) dominant negative mutant ERK1 and Smad3 constructs decrease TGF-beta1-induced collagen I gene promoter activity. From these data, we propose the hypothesis that both the MAP kinase and SMAD pathways are activated by TGF-beta1 in human mesangial cells and are relevant to matrix accumulation. We will test this hypothesis via four specific aims. First, we will characterize TGF-beta1 activation of the ERK and JNK MAP kinase, and SMAD, pathways, evaluating protein phosphorylation, nuclear translocation and possible interaction between the two pathways. Second, we will determine whether interrupting the SMAD or MAP kinase pathways prevents induction of expression or activation of gene promoters for type I and type IV collagen by TGF- beta1. Third, we will examine the mechanism of activation of collagen genes by identifying TGF-beta1-responsive elements in the gene promoters and determining whether MAP kinases and/or SMADs participate in transcription-regulatory complexes that bind to these DNA sequences. Finally, we will seek to relate our findings to glomerulosclerosis by determining whether SMADs and MAP kinases are expressed and activated in human disease or in animal models of glomerular collagen accumulation. These studies will provide a model for cell signaling mechanisms that mediate matrix accumulation by mesangial cells and may suggest means for altering abnormal matrix turnover in glomerulosclerosis.